The long-term goal of our research is to understand the structural basis of the complex processes that regulate the replication cycle of rotaviruses (RVs), which are the global pathogens causing life-threatening infantile gastroenteritis. During 2013-2016, our studies have answered several questions we previously asked, raised a new set of questions, and revealed novel concepts. As a result, we plan to pursue exciting new directions during the MERIT extension period (2018-2023) through four new AIMS. In pursuing these AIMs, we will use a multipronged approach involving glycan array screening, X-ray crystallography, single-particle cryo-EM, cryo-electron tomography of RV-infected cells, and functional assays. In AIM1, considering that specific recognition of host cell glycans is a critical factor in host cell attachment and cross-species transmission, we will address new questions such as: (i) what is the glycan specificity in sialidase-insensitive animal RVs with zoonotic potential?; (ii) do these viruses show similar correlated glycan specificity with the human RVs we discovered in bovine and human P[11] RVs to cross the species barrier?; (iii) does glycan binding affect spike structure in the intact virion to influence downstream cell entry processes?; and (iv) do VP8*-specific human mAbs block glycan binding in human RVs?. In AIM 2, our goal is to understand the structural aspects of the viral capping enzyme VP3, how it associates with the viral polymerase VP1 to gain insight into the mechanistic basis of endogenous transcription, and the possible role of VP3 in capsid assembly and genome encapsidation that occurs in the specialized replication factories called viroplasms. Experiments in AIM 3 are designed to probe further into understanding protein-protein interaction networks that regulate viroplasm-associated activities using structural techniques and cryo-ET of RV-infected cells at different time points post infection. In AIM 4, our goal is to provide structure-based mechanistic insights into how RVs antagonize cellular antiviral responses by understanding structural aspects of RV proteins such as NSP1 that inhibit IFN pathways and the phosphodiesterase domain embedded in VP3 of group-A RVs that inactivates OAS/RNase-L pathway, and the dsRNA-binding domain in NSP3 of group-C RVs that inhibits dsRNA-dependent protein kinase.